The plasma membrane of eukaryotic cells is a tightly controlled barrier protecting the cell from unregulated influx of bioactive molecules. For small-molecule and protein or other macromolecule-based drugs that are not endogenous to the cell, traversing the plasma membrane can involve either using a natural transport system or achieving direct diffusion through the lipid bilayer. However, in many cases, both modes of entry are inefficient for exogenous molecules.
In the past decade, cell penetrating peptides (CPPs) have emerged as promising vectors to deliver different therapeutic agents, including proteins, to their targets. CPPs are peptide sequences facilitating efficient protein translocation across biological membranes, independently of transporters or specific receptors. CPPs also offer the advantage of cost-efficient production. Since the discovery 20 years ago of the membrane translocating property of human immunodeficiency virus transactivating regulatory protein (HIV TAT), several CPPs have been identified including penetratin (Antennapedia homeodomain), VP22 (Herpes simplex virus) and the synthetic polyarginine (polyR). Different cargoes have been linked to CPPs with the perspective of novel vaccine design.
Recently, the Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. More particularly, it has been demonstrated that the minimal region of ZEBRA, which is necessary for the cell-penetrating ability of the Epstein-Barr virus ZEBRA trans-activator, spans from residue 170 to residue 220 of ZEBRA and that both the DNA binding domain and the dimerization domain contained in that region are necessary for the cell-penetrating properties (Rothe et al. 2010, J. Biological Chemistry 285(26): 20224-20232).
Many CPPs have drawbacks when their use as vehicles for delivery of cargo molecules into the cell is considered. For instance, they can cause severe side-effects in the cell such as cytoplasmic leakage due to membrane disruption or interference with the normal functioning of membrane proteins, or can cause cellular toxic effects and/or immunogenic effects. Also, some CPPs may be rapidly degraded in the cytoplasm or remain entrapped in endosomes to be degraded in lysosomes. Moreover, some CPPs are unable to release the cargo molecule, or do not have a broad spectrum for addressing or releasing the cargo molecule.
Therefore, there is still a need for an improved vehicle able to deliver a wide variety of cargo molecules into a cell, which exhibits a high efficiency of uptake of the cargo molecule as well as low toxicity. In the context of vaccines, it would also be advantageous if the delivery vehicle was not restricted to only one pathway for internalization of the cargo molecule and could be delivered to both the cytosol and endosomes for antigen presentation. The present invention solves this problem by providing CPPs which allow efficient delivery and presentation of, for instance, antigenic determinants at the cell surface of antigen presenting cells. The CPPs of the invention, thus, are useful as vehicles for delivery of a variety of cargo molecules such as polypeptides and proteins, polysaccharides, lipids, or combinations thereof, as well as nucleic acids, small molecule drugs, and imaging agents.